Crystal structures of rice hexokinase 6 with a series of substrates shed light on its enzymatic mechanism.

Hexokinases (HXKs) have determined to be multifaceted proteins, and they are the only ones able to phosphorylate glucose in plants. However, the binding mode for ATP to plant HXKs remains unclear. Here, we report the crystal structures of rice hexokinase 6 (OsHXK6) in four different forms: (i) apo-form, (ii) binary complex with D-Glc, (iii) quaternary complex with ADP, PO4 and Mg2+, and (iv) pentanary complex with D-Glc, ADP, PO4, and Mg2+. The apo form is in the open state conformation, and the three others are in the closed state, indicating that glucose and ADP-PO4 binding induces a large conformational change by domain rearrangement. The quaternary complex is a novel intermediate during the catalytic reaction we trapped for the first time, which provides a new evidence for the enzymatic mechanism of HXKs. In addition, the latter two complexes reveal the binding mode for ADP-PO4 to plant HXKs, which provide the structural explanation for the dual-function of OsHXK6. In addition, we identified that residues Gly112, Thr261, Gly262, and Gly450 are essential to the binding between ADP-PO4 and OsHXK6 by a series of single mutations and enzymatic assays. Our study provide structural basis for the other functional studies of OsHXK6 in rice.

A metabolome-based core hybridisation strategy for the prediction of rice grain weight across environments.

Marker-based prediction holds great promise for improving current plant and animal breeding efficiencies. However, the predictabilities of complex traits are always severely affected by negative factors, including distant relatedness, environmental discrepancies, unknown population structures, and indeterminate numbers of predictive variables. In this study, we utilised two independent F1 hybrid populations in the years 2012 and 2015 to predict rice thousand grain weight (TGW) using parental untargeted metabolite profiles with a partial least squares regression method. A stable predictive model for TGW was built based on hybrids from the population in 2012 (r = 0.75) but failed to properly predict TGW for hybrids from the population in 2015 (r = 0.27). After integrating hybrids from both populations into the training set, the TGW of hybrids could be predicted but was largely dependent on population structures. Then, core hybrids from each population were determined by principal component analysis and the TGW of hybrids in both environments were successfully predicted (r > 0.60). Moreover, adjusting the population structures and numbers of predictive analytes increased TGW predictability for hybrids in 2015 (r = 0.72). Our study demonstrates that the TGW of F1 hybrids across environments can be accurately predicted based on parental untargeted metabolite profiles with a core hybridisation strategy in rice. Metabolic biomarkers identified from early developmental stage tissues, which are grown under experimental conditions, may represent a workable approach towards the robust prediction of major agronomic traits for climate-adaptive varieties.

Identification of hepatic fibroblast growth factor 21 as a mediator in 17ß-estradiol-induced white adipose tissue browning.

Both ovarian E2 and hepatic fibroblast growth factor 21 (FGF21) are critical for energy homeostasis and white adipose tissue browning. Estrogen receptor α (ERα) is abundantly expressed in liver. However, whether FGF21 has a role in E2-induced white adipose tissue browning remains uncertain. In this study, we showed that hepatic Fgf21 expression and secretion during estrus cycle changed with the tetradian oscillatory secretion of circulation E2 in adult, female mice, with their peak expressions and secretions at the proestrus. In addition, exogenous E2 robustly stimulated liver Fgf21 expression and elevated serum FGF21 concentrations, which induced browning gene expression and reduced the tissue weight in subcutaneous white adipose in mice with ovariectomies. The inhibitor of mammalian target of rapamycin (mTOR) and of ERα blocked the induction effect of E2 on the expression of Fgf21 in primary hepatocytes, which revealed that E2 might stimulate FGF21 expression via the ERα-mTOR pathway. Furthermore, FGF21 liver-specific deficiency abolished E2-induced white adipose browning in mice with ovariectomies. This study indicates that ovarian E2 increased liver FGF21 expression directly, which in turn, functioned as an endocrine signal to influence inguinal white adipose tissue browning.-Hua, L., Zhuo, Y., Jiang, D., Li, J., Huang, X., Zhu, Y., Li, Z., Yan, L., Jin, C., Jiang, X., Che, L., Fang, Z., Lin, Y., Xu, S., Li, J., Feng, B., Wu, D. Identification of hepatic fibroblast growth factor 21 as a mediator in 17ß-estradiol-induced white adipose tissue browning.

Rice PPS1 encodes a DYW motif-containing pentatricopeptide repeat protein required for five consecutive RNA-editing sites of nad3 in mitochondria.

The pentatricopeptide repeat (PPR) protein family is a large family characterized by tandem arrays of a degenerate 35-amino-acid motif whose members function as important regulators of organelle gene expression at the post-transcriptional level. Despite the roles of PPRs in RNA editing in organelles, their editing activities and the underlying mechanism remain obscure. Here, we show that a novel DYW motif-containing PPR protein, PPS1, is associated with five conserved RNA-editing sites of nad3 located in close proximity to each other in mitochondria, all of which involve conversion from proline to leucine in rice. Both pps1 RNAi and heterozygous plants are characterized by delayed development and partial pollen sterility at vegetative stages and reproductive stage. RNA electrophoresis mobility shift assays (REMSAs) and reciprocal competition assays using different versions of nad3 probes confirm that PPS1 can bind to cis-elements near the five affected sites, which is distinct from the existing mode of PPR-RNA binding because of the continuity of the editing sites. Loss of editing at nad3 in pps1 reduces the activity of several complexes in the mitochondrial electron transport chain and affects mitochondrial morphology. Taken together, our results indicate that PPS1 is required for specific editing sites in nad3 in rice.

A rice dual-localized pentatricopeptide repeat protein is involved in organellar RNA editing together with OsMORFs.

In flowering plants, various RNA editing events occur in the mitochondria and chloroplasts as part of post-transcriptional processes. Although several pentatricopeptide repeat (PPR) proteins and multiple organellar RNA editing factors (MORFs) have been identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among these proteins are still obscure. Here, we identified a rice dual-localized PPR protein, OsPGL1. The loss of function of OsPGL1 resulted in defects in both chloroplast RNA editing of ndhD-878 and mitochondrial RNA editing of ccmFc-543, both of which could be restored in transgenic complementation lines. Despite synonymous editing of ccmFc-543, the loss of editing of ndhD-878 caused a failed conversion of serine to leucine, leading to chloroplast dysfunction and defects in the photosynthetic complex; the results of additional experiments demonstrated that OsPGL1 directly binds to both transcripts. Interactions between three OsMORFs (OsMORF2/8/9) and OsPGL1 both in vitro and in vivo were confirmed, implying that OsPGL1 functions in RNA editing via an editosome. These findings also suggested that OsMORFs assist with and contribute to a flexible PPR-RNA recognition model during RNA editing. These results indicate that, in cooperation with PPRs, OsPGL1 is required for RNA editing. In addition, our study provides new insights into the relationship between RNA editing and plant development.

Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.

The rice DUF1620-containing and WD40-like repeat protein is required for the assembly of the restoration of fertility complex.

Cytoplasmic male sterility (CMS) and restoration of fertility (Rf) are widely distributed in plant species utilized by humans. RF5 and GRP162 are subunits of the restoration of fertility complex (RFC) in Hong-Lian rice. Despite the fact that the RFC is 400-500 kDa in size, the other proteins or factors in the complex still remain unknown. Here, we identified RFC subunit 3, which encodes a DUF1620-containing and WD40-like repeat protein (RFC3) that is present in all tissues but highly expressed in leaves. We established that RFC3 interacts with both RF5 and GRP162 in vitro and in vivo, and is transported into the mitochondria as a membrane protein. Furthermore, CMS RNA (atp6-orfH79) and CMS cytotoxic protein (ORFH79) accumulate when RFC3 is silenced in restorer lines. We presented the analysis with blue-native polyacrylamide gel electrophoresis, indicating that RFC is disrupted in the RNAi line. We concluded that RCF3 is indispensable as a scaffold protein for the assembly of the RFC complex. We unveil a new molecular player of the RFC in the Rf pathway in rice and propose the model of RFC based on these data.

OsDi19-4 acts downstream of OsCDPK14 to positively regulate ABA response in rice.

The drought-induced 19 protein family consists of several atypical Cys2/His2-type zinc finger proteins in plants and plays an important role in abiotic stress. In this study, we found that overexpressing OsDi19-4 in rice altered the expression of a series of abscisic acid (ABA)-responsive genes, resulting in strong ABA-hypersensitive phenotypes including ABA-induced seed germination inhibition, early seedling growth inhibition and stomatal closure. On the contrary, OsDi19-4 knockdown lines were less sensitive to ABA. Additionally, OsCDPK14 was identified to interact with OsDi19-4 and be responsible for the phosphorylation of OsDi19-4, and the phosphorylation of OsDi19-4 was further enhanced after the treatment of ABA. Apart from these, OsDi19-4 was shown to directly bind to the promoters of OsASPG1 and OsNAC18 genes, two ABA-responsive genes, and regulate their expression. Transient expression assays confirmed the direct regulation role of OsDi19-4, and the regulation was further enhanced by the increased phosphorylation of OsDi19-4 after the treatment of ABA. Taken together, these data demonstrate that OsDi19-4 acts downstream of OsCDPK14 to positively regulate ABA response by modulating the expression of ABA-responsive genes in rice.

Global epigenomic analysis indicates that epialleles contribute to Allele-specific expression via Allele-specific histone modifications in hybrid rice.

BACKGROUND: For heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants. RESULTS: We used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study. CONCLUSIONS: Our results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice.

Identification of tapetum-specific genes by comparing global gene expression of four different male sterile lines in Brassica oleracea.

The tapetum plays an important role in anther development by providing necessary enzymes and nutrients for pollen development. However, it is difficult to identify tapetum-specific genes on a large-scale because of the difficulty of separating tapetum cells from other anther tissues. Here, we reported the identification of tapetum-specific genes by comparing the gene expression patterns of four male sterile (MS) lines of Brassica oleracea. The abortive phenotypes of the four MS lines revealed different defects in tapetum and pollen development but normal anther wall development when observed by transmission electron microscopy. These tapetum displayed continuous defective characteristics throughout the anther developmental stages. The transcriptome from flower buds, covering all anther developmental stages, was analyzed and bioinformatics analyses exploring tapetum development-related genes were performed. We identified 1,005 genes differentially expressed in at least one of the MS lines and 104 were non-pollen expressed genes (NPGs). Most of the identified NPGs were tapetum-specific genes considering that anther walls were normally developed in all four MS lines. Among the 104 NPGs, 22 genes were previously reported as being involved in tapetum development. We further separated the expressed NPGs into different developmental stages based on the MS defects. The data obtained in this study are not only informative for research on tapetum development in B. oleracea, but are also useful for genetic pathway research in other related species.

Mitochondrial ORFH79 is Essential for Drought and Salt Tolerance in Rice.

The mitochondrion is deemed to be one of the most important organelles, and plays an essential role in various biological processes. Nonetheless, the role of mitochondria in response to abiotic stress remains unclear. Here, we report that accumulation of the cytoplasmic male sterility (CMS) protein ORFH79 in the vegetative tissues resulted in the dysfunction of mitochondria with decreased enzymatic activities of respiratory chain complexes, reduced ATP content and even a morphological change of the mitochondria. However, the suppression of orfH79 by overexpressing a fertility restorer gene Rf5, which is targeted to mitochondria and induced an endonucleolytic cleavage on the atp6-orfH79 transcripts, could recover the function of mitochondria and further significantly improved the tolerance to drought and salt stress. The above evidence suggests that the mitochondrion plays a pivotal role in tolerance to drought and salt stress in rice.

The rice pentatricopeptide repeat protein RF5 restores fertility in Hong-Lian cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162.

The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene-mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS-Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration.

The phenotypic predisposition of the parent in F1 hybrid is correlated with transcriptome preference of the positive general combining ability parent.

BACKGROUND: Sprague and Tatum (1942) introduced the concepts of general combining ability (GCA) and specific combining ability (SCA) to evaluate the breeding parents and F1 hybrid performance, respectively. Since then, the GCA was widely used in cross breeding for elite parent selection. However, the molecular basis of GCA remains to unknown. RESULTS: We studied the transcriptomes of three varieties and three F1 hybrids using RNA-Sequencing. Transcriptome sequence analysis revealed that the transcriptome profiles of the F1s were similar to the positive GCA-effect parent. Moreover, the expression levels of most differentially expressed genes (DEGs) were equal to the parent with a positive GCA effect. Analysis of the gene expression patterns of gibberellic acid (GA) and flowering time pathways that determine plant height and flowering time in rice validated the preferential transcriptome expression of the parents with positive GCA effect. Furthermore, H3K36me3 modification bias in the Pseudo-Response Regulators (PRR) gene family was observed in the positive GCA effect parents and demonstrated that the phenotype and transcriptome bias in the positive GCA effect parents have been epigenetically regulated by either global modification or specific signaling pathways in rice. CONCLUSIONS: The results revealed that the transcriptome profiles and DEGs in the F1s were highly related to phenotype bias to the positive GCA-effect parent. The transcriptome bias toward high GCA parents in F1 hybrids attributed to H3K36me3 modification both on global modification level and specific signaling pathways. Our results indicated the transcriptome profile and epigenetic modification level bias to high GCA parents could be the molecular basis of GCA.

Protoplast: a more efficient system to study nucleo-cytoplasmic interactions.

Agrobacterium tumefaciens-mediated genetic transformation is a powerful tool for plant research, but it can be labor-intensive and time-consuming. Here, we report a protoplast-based approach to study nucleo-cytoplasmic interactions, such as cytoplasmic male sterility/fertility restoration (CMS/Rf) and organellar RNA editing. To test the system, we transfected the fertility restorer gene Rf5, which is involved in the rice HL-CMS/Rf system, into rice protoplasts prepared from the HL-CMS line. As the Rf5 protein accumulated in the transformed protoplasts, the CMS-associated transcripts were endonucleolytically cleaved. There were much lower levels of the CMS-associated protein ORFH79 in the transfected protoplasts than in the mock-transfected protoplasts. Next, we used a dsRNA-mediated gene silencing approach to down-regulate the pentatricopeptide protein gene MPR25, which participates in RNA editing of the organellar transcript nad5. The editing efficiency of mitochondrial transcripts of nad5 at nucleotide 1580 was much lower in the transfected protoplasts than in the mock-transfected protoplasts. Together, these results show that protoplast is a simple and efficient system to study interactions between the nucleus and organelles.

Interaction with Cu²âº disrupts the RNA binding affinities of RNA recognition motif containing protein.

The glycine-rich proteins (GRP) containing RNA recognition motifs (RRM) are involved in the regulation of transcriptional and/or post-transcriptional events. Previous studies have established that GRP162 plays an important role in the restoration of fertility in Honglian cytoplasmic male sterile (HL-CMS) rice. In this study, the ion binding properties of rGRP162 were tested by isothermal titration calorimetry (ITC) and electrophoretic mobility shift assay (EMSA) was performed to test the interaction. Circular dichroism (CD) was carried out to detect the alteration of secondary structure in the presence and absence of Cu(2+). Furthermore, two RRM containing proteins, AtRBP45A and AtRBP47A, were expressed to validate the interaction. Results showed Cu(2+) and Fe(3+) bound GRP162, whereas Ca(2+), Mn(2+), Mg(2+) and K(+) did not. EMSA confirmed that interaction with Cu(2+) interrupted the biological activity of GRP162 by disrupting the secondary structure of the protein based on the results of CD. Moreover, the RNA binding activities of rAtRBP45A and rAtRBP47A were also impaired in the presence of Cu(2+). Data suggest that Cu(2+) in excess may disrupt RNA-binding proteins containing RRM that are essential for post-transcriptional regulation and may impair the development of plants or animals.

Identification of rice Di19 family reveals OsDi19-4 involved in drought resistance.

KEY MESSAGE: The OsDi19 proteins functioned as transcription factors and played crucial roles in response to abiotic stress. Overexpression of OsDi19-4 in rice increased drought tolerance by enhancing ROS-scavenging activity. Many transcription factors play crucial roles in plant responses to abiotic stress. Here, comprehensive sequence analysis suggested that the drought-induced 19 (Di19) gene family in rice genome contain seven members, and these proteins contained a well-conserved zinc-finger Di19 domain. Most OsDi19 proteins were mainly targeted to the nucleus and have transactivation activity in yeast. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that most OsDi19 proteins could form protein dimers. Expression analysis demonstrated that the OsDi19 genes were differentially and abundantly expressed in vegetative tissues, but expressed little in reproductive tissues and some of the OsDi19 genes were markedly induced by abiotic stresses and hormones in qRT-PCR analysis and microarray data. Overexpression of one stress-responsive gene, OsDi19-4, in rice resulted in significantly increased tolerance to drought stress compared with the wild type plants. Moreover, obviously increased ROS-scavenging ability was detected in the OsDi19-4-overexpressing plants under normal and drought stress conditions. These results suggested that the increased stress tolerance of OsDi19-4-overexpressing plants may be attributable to the enhanced ROS-scavenging activity. Taken together, these studies provide a detailed overview of the rice Di19 gene family, and suggest that the OsDi19 family may play crucial roles in the plant response to abiotic stress.

Defining reference genes for quantitative real-time PCR analysis of anther development in rice.

Quantitative real-time polymerase chain reaction (qPCR) is one of the most accurate and widely used methods for gene expression analysis. However, the choice of reference genes for normalization is critical for accurate quantification of gene expression. As development of genomics, mining large-scale datasets such as microarray and RNA-sequencing data becomes a new approach for exploitation of new reference genes. In this study, we analyzed an RNA-sequencing dataset of rice anther and 167 microarray datasets involving different tissues and developing stages of rice anthers and pollens. We selected 12 candidate genes and other 5 reference genes, including ACT1, eEF-1α, GAPDH, Exp2, and CCDC72 used in previous studies, and evaluated their expression in eight tissues and different developmental stages of anthers in rice variety 9311 and Yuetai. UPF3, eIF4A-3, GAPDH, and PPP6 were identified as the most suitable reference genes for qPCR analysis of anther development in rice. The new candidate reference genes showed more stable expression than the traditionally used reference genes. These results provide a set of reliable reference genes for studies in rice anther developmental process.

Large-scale production of functional human serum albumin from transgenic rice seeds.

Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.

Overexpression of OsKTN80a, a katanin P80 ortholog, caused the repressed cell elongation and stalled cell division mediated by microtubule apparatus defects in primary root in Oryza sativa.

Katanin, a microtubule-severing enzyme, consists of two subunits: the catalytic subunit P60, and the regulatory subunit P80. In several species, P80 functions in meiotic spindle organization, the flagella biogenesis, the neuronal development, and the male gamete production. However, the P80 function in higher plants remains elusive. In this study, we found that there are three katanin P80 orthologs (OsKTN80a, OsKTN80b, and OsKTN80c) in Oryza sativa L. Overexpression of OsKTN80a caused the retarded root growth of rice seedlings. Further investigation indicates that the retained root growth was caused by the repressed cell elongation in the elongation zone and the stalled cytokinesis in the division zone in the root tip. The in vivo examination suggests that OsKTN80a acts as a microtubule stabilizer. We prove that OsKTN80a, possibly associated with OsKTN60, is involved in root growth via regulating the cell elongation and division.

Alterations of mitochondrial protein assembly and jasmonic acid biosynthesis pathway in Honglian (HL)-type cytoplasmic male sterility rice.

It has been suggested that the mitochondrial chimeric gene orfH79 is the cause for abortion of microspores in Honglian cytoplasmic male sterile rice, yet little is known regarding its mechanism of action. In this study, we used a mass spectrometry-based quantitative proteomics strategy to compare the mitochondrial proteome between the sterile line Yuetai A and its fertile near-isogenic line Yuetai B. We discovered a reduced quantity of specific proteins in mitochondrial complexes in Yuetai A compared with Yuetai B, indicating a defect in mitochondrial complex assembly in the sterile line. Western blotting showed that ORFH79 protein and ATP1 protein, an F(1) sector component of complex V, are both associated with large protein complexes of similar size. Respiratory complex activity assays and transmission electron microscopy revealed functional and morphological defects in the mitochondria of Yuetai A when compared with Yuetai B. In addition, we identified one sex determination TASSELSEED2-like protein increased in Yuetai A, leading to the discovery of an aberrant variation of the jasmonic acid pathway during the development of microspores.